JP5745515B2 - Method for detection of kidney damage - Google Patents

Description

Detailed Description of the Invention

  The present invention relates to a method for determining the presence of renal damage in an individual, which also includes Reg3B, fetuin B, Ras-related GTP binding protein A, serine protease inhibitor A3L, COP9 subunit 1, ATP. Detect one or several proteins selected from a list including the gamma subunit of synthase, gelsolin, ribonuclease UK114, aminoacylase 1A, alpha enolase, keratin 5, parvalbumin alpha, ribonuclease 4, or serine protease inhibitor A3K Related to the method. Renal injury can be acute renal failure. This renal pathology can be caused by administration of a nephrotoxic factor, wherein the nephrotoxic factor can be an aminoglycoside antibiotic (eg, gentamicin or cisplatin). The present invention also provides a means to differentiate renal damage or renal failure caused by gentamicin from that caused by cisplatin through biochemical analysis of urinary levels of Reg3B and / or gelsolin, or fragments thereof. provide.

[Background Technology]
Aminoglycoside antibiotics are widely used against bacterial infections. In particular, gentamicin is used against gram-negative infections. Its therapeutic use and efficacy is considerably hampered by its toxicity and occurs mainly at the renal and auditory levels (Martinez-Salgado C, Lopez-Hernandez FJ and Lopez-Novoa JM. 2007, Toxicol Appl Pharmacol., 223: 86 -98). Gentamicin-induced nephrotoxicity is seen in 10-25% of treatments (Leehey DJ et al., 1993, J. Am. Soc. Nephrol., 4: 81-90). This is mainly characterized by tubular damage (Nakakuki M et al., 1996, Can J Physiol Pharmacol., 74: 104-111). However, glomeruli (Martinez-Salgado C, Lopez-Hernandez FJ and Lopez-Novoa JM. 2007, Toxicol Appl Pharmacol., 223: 86-98) and vessels (Goto T et al., 2004, Virchows Arch., 444 : 362-74; Secilmis MA, et al., 2005, Nephron Physiol., 100: 13-20) can also appear in a dose-dependent manner (Hishida A et al., 1994, Ren Fail. , 16: 109-116). Tubular damage primarily affects the proximal tubule, and depending on the intensity of the attack, the lesion can range from mild scaling to more severe tubular necrosis (Nakakuki et al. al., 1996. Can J Physiol Pharmacol., 74: 104-111). Tubular lesions cause an imbalance in the ability of reabsorption to activate tubular glomerular feedback and dramatically reduce glomerular filtration rate (GFR) to prevent loss of large amounts of fluid. Finally, gentamicin reduces renal blood flow (RBF) by contraction of the anterior glomerular artery and afferent and efferent arterioles (Klotman et al., 1983. Kidney Int., 24: 638-643 ). As a result of the decrease in RBF, GFR deteriorates. Decreased RBF also results in tissue necrosis, particularly inside the cortex (Cheung et al., 2008. Drugs Aging, 25: 455-476).

  Gentamicin-related occupational toxicity is occasionally associated with acute renal failure, depending on age, gender, previous renal function, treatment organ, treatment regimen, hydration level, and other associated conditions (eg, pregnancy or hypothyroidism) Can be reached.

  Acute renal failure is a type of kidney injury characterized by a loss of renal excretory function and inhibits blood waste and water purification and maintenance of electrolyte balance (Bellomo R, Kellum JA and Ronco C, 2007). Intensive Care Med., 33: 409-413). Acute kidney injury and acute renal failure can be induced by a wide range of attacks (eg, drugs, chemical poisons, hypoxia, renal urinary tract disorders, infections, etc.) (Binswanger U, 1997. Kidney Blood Press Res., 20: 163). This type of kidney injury represents a huge burden on humans and socio-economics due to its high incidence and mortality. It is estimated that about 1% of patients presenting are associated with acute renal failure, and about 2-7% of hospitalized patients eventually exacerbate this. More importantly, the mortality rate of patients with acute renal failure is still significantly higher, about 50% of cases.

  In the clinical setting, acute renal failure (and in general acute kidney injury) is diagnosed when renal dysfunction produces detectable symptoms. These are typically based on measuring creatinine and urea levels. Most commonly, these serum concentrations increase as GFR decreases. However, acute renal failure is difficult to treat if high serum levels of urea and creatinine have already been observed. Thus, the current epidemic in diagnosis is aimed at detecting the occurrence of physiopathological events occurring at an early stage when the injury is localized (Vaidya VS, Ferguson MA and Bonventre JV, 2008. Rev Pharmacol Toxicol., 48: 463-493). Of these, the measurement of specific cellular enzymes present in urine as a result of tubular cell disruption is currently the most sophisticated for early detection of acute kidney injury that occurs with tubular injury It is a procedure. These enzymes include N-acetyl-D-glucosaminidase (NAG), but also include, for example, lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and γ-glutamyl transpeptidase (GGT). Most of these enzymes have moderate value as early and sensitive urinary markers of acute kidney injury due to stability and inhibition problems with other urine components (Vaidya VS, Ferguson MA and Bonventre JV, 2008. Rev Pharmacol Toxicol., 48: 463-493). In addition to the above, the latest generation of early markers includes urine measurement of renal injury molecule 1 (KIM-1) or neutrophil gelatinase-related lipocalin (NGAL) (Vaidya et al., 2008).

  However, there are still aspects that can be improved in the diagnosis of acute kidney injury and acute renal failure. One of these aspects is an etiological diagnosis of damage, in other words, a diagnosis that not only detects damage early but also detects its cause. This aspect becomes particularly important in clinical situations where potentially different nephrotoxic factors, such as various drugs that are potentially harmful to the kidneys, are simultaneously concentrated on the same individual. Under these circumstances, when the first symptoms of nephrotoxicity appear, it is impossible to know what caused the damage using the current diagnostic techniques, and consequently Preventing the replacement or alteration of a drug-only treatment regimen without altering the other drug treatment regimen. Among these types of drugs, aminoglycoside antibiotics have attracted attention. The ability to identify a specific marker or set of markers in this manner is more rational, individual and specific, such as simultaneous treatment involving two or more potential nephrotoxic drugs in daily clinical situations Makes it possible to provide treatment. This allows treatment to be directed correctly by replacing or changing the treatment regimen of harmful drugs.

[Summary of the Invention]
The present invention relates to a method for determining renal damage in an individual, as well as Reg3B, fetuin B, Ras-related GTP binding protein A, serine protease inhibitor A3L, COP9 subunit 1, γ subunit of ATP synthase, gelsolin, ribonuclease Predicting the progression of gin damage by detecting one or several proteins selected from the list including UK114, aminoacylase 1A, alpha enolase, keratin 5, parvalbumin alpha, ribonuclease 4, or serine protease inhibitor A3K Related to the method. Renal injury can be acute renal failure. This renal pathology can be caused by administration of a nephrotoxic factor, wherein the nephrotoxic factor can be an aminoglycoside antibiotic (eg, gentamicin or cisplatin).

  The present invention provides evidence related to increased excretion of any protein or any combination thereof selected from the list above, where kidney damage or acute renal failure induced by non-limiting exemplary gentamicin.

  Thus, the present invention provides a tool for detecting renal injury or acute renal failure. These tools allow the progression of renal injury or acute renal failure to be predicted, in other words, when a human is treated with a therapeutic agent as a non-limiting example, or when a human is The progression of the pathology is monitored when exposed to nephrotoxic or non-nephrotoxic factors or situations.

  The present invention also includes Reg3B, fetuin B, Ras-related GTP-binding protein A, serine protease inhibitor A3L, COP9 subunit 1, ATP synthase gamma subunit, gelsolin, ribonuclease UK114, aminoacylase 1A, α enolase, keratin 5 Provides a significant advantage of detecting in a urine sample a protein selected from the list comprising parvalbumin alpha, ribonuclease 4, or serine protease inhibitor A3K, or any fragment thereof, and the excretion of body fluids is a natural occurrence This necessarily entails additional benefits for the patient. This means that sampling from individuals is not aggressive.

  Ultimately, the present invention provides a means to detect and differentiate whether renal injury or renal failure has been caused by gentamicin or cisplatin. This is another diagnostically significant advantage that distinguishes the factors of renal injury or renal failure in order to properly reconstruct the treatment in patients who are prescribed many drugs.

  A brief description of the proteins detected and / or quantified in the method of the present invention is as follows.

  Regenerating islet-derived protein 3 beta (REG3β, REG-III or RegIII β) is also known as pancreatic stone protein 2, known as pancreatic-associated protein 1 (Pap), Pancreatic-associated protein 1 (Pap) These are synonymous. This protein is related to lectins. Reg3B is present in small amounts in normal pancreas but is overexpressed in the acute phase of pancreatitis. This may be related to the response to control bacterial growth during acute pancreatitis. It is possible to clone this.

  Fetuin B (otherwise known as 16G2, fetuin B precursor, Gugu, IRL685) is a protein synthesized in the kidney and secreted into the bloodstream. It belongs to a large group of proteins that facilitate the transport and availability of a wide variety of substances in the bloodstream. This protein is more ubiquitous in fetal blood than in the blood flow of adult individuals, and thus has been termed “fetuin”.

  Ras-related GTP binding protein A (also known as Rag A, FIP1, FIP-1, RagA, RAGA, RRAGA, Rag A, or adenovirus E3 14.7 kDa-interacting protein 1) is a homodimer. It can be in the form of This is related to the Rcc1 / Ran-GTPase signaling pathway and may exert a direct function in TNFα-mediated signaling leading to the induction of cell death. It is ubiquitously expressed in skeletal muscle, heart and brain.

  The serpin A3L protein (which is referred to as serine protease inhibitor A3L, serine protease inhibitor 1, or contratrypsin-like protease inhibitor 3) is a group of proteins that can inhibit other enzymes in the protease group. Belongs. The name Serpin comes from a combination of serine proteases in terms of their functional properties. Seprin A3L is induced by growth hormone and has been described to reduce its expression level during acute inflammation in rats. The expression product of this gene is localized in the liver of Rattus novelgicus (rat). This seprin A3K protein (also referred to as contratrypsin-like protease inhibitor 1, kallikrein-binding protein, serine protease inhibitor 2, or growth hormone-regulated protease inhibitor) is an extracellular seprin protein and is the retina of rats with diabetes. At low levels and may contribute to retinopathy.

  Signalsome COP9 is a protein complex conserved in eukaryotic cells, and is composed of eight subunits (CSN1 to CSN8). COP9 is a ubiquitination regulator. Ubiquitination consists of the process of marking proteins with ubiquitin proteins for proteolysis. Ubiquitin anchors to the protein to be removed, and in this manner, the marked protein moves to the proteosome, the structure where the proteolytic process takes place.

  The γ subunit of ATP synthase (also known as the γ subunit of F type ATPase) forms the central axis that links the F0 rotary domain of this complex to the F1 catalytic domain. ATP synthase protein uses ADP to produce ATP in the presence of a proton gradient between inside and outside the cell. F type ATPase has two components. The F1 component has a catalytic function, and the F0 component is a proton channel embedded in the membrane. F1 has five subunits (α, β, γ, δ and ε). F0 has three major subunits (a, b and c). The γ subunit is important in regulating complex activity and proton flux.

  Gelsolin is a 82 kDa globular protein with six subunits called S1-S6. This protein is associated with the assembly of actin filaments.

  Ribonuclease (RNase) is an enzyme (nuclease) that catalyzes the hydrolysis of RNA into smaller components. Ribonuclease UK114 is responsible for translational inhibition and mRNA hydrolysis. Ribonuclease 4 (RNase 4) is a protein of about 16 kDa that favors hydrolysis of ribonucleic acid polymers.

  The enzyme aminoacylase 1 (Acy1) is a homodimer that is localized in the cytoplasm and relies on its zinc bond and catalyzes the hydrolysis of acylated L amino acids.

  Alpha enolase (well known as a glycolytic enzyme) has been identified as a self-antigen in Hashimoto encephalopathy and is associated with severe asthma. A decrease in the expression of this enzyme is found in the cornea of humans suffering from keratoconus.

  Keratin 5 is a cytokeratin often associated with keratin 14. Type II cytokeratins are composed of basic or neutral proteins, organized into a pair of chains, and differ from keratins that are co-expressed during differentiation of simple and layered epithelial tissues.

  Parvalbumin protein is a low molecular weight albumin (usually 9 to 11 kDa) and needs to bind to calcium in order to exert its function. This is structurally related to calmodulin and troponin C. Parvalbumin is localized in fast contracting muscles, brain and some secretory tissues.

One aspect of the present invention relates to a method for providing data useful for determining renal injury, the method comprising:
a. Obtaining an isolated biological sample from an individual; and b. In the obtained sample,
A protein having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or any fragment thereof;
A protein having at least 80% identity to the amino acid sequence of SEQ ID NOs: 3-11, or any fragment thereof; or a protein having at least 90% identity to the amino acid sequence of SEQ ID NOs: 12-14, or any fragment thereof Detecting and / or quantifying at least one or any combination of proteins selected from a list comprising:

  As used herein, the term “renal injury” refers to any damage in the renal urinary system, which may or may not cause a condition in an individual that results in kidney disease. . The origin of kidney damage includes, but is not limited to, genetic, immune system, ischemia, or treatment with any drug. Renal injury or the diseases described above can also be generated by non-limiting exemplary kidney, prostate, bladder, ureter or urethral surgical procedures.

The following describes the correspondence between the referenced sequence and the described protein, and lists each accession number and the organism from which it is derived:
-Sequence number 1; Reg3B (A49616; corresponding to Homo sapiens)
SEQ ID NO: 2; fetuin B (NP_055190.2; corresponding to Homo sapiens)
SEQ ID NO: 3; serine protease inhibitor A3K (P05545.3; corresponding to Rattus norvegicus)
SEQ ID NO: 4; γ subunit of ATP synthase (NP_001001973.1; corresponding to Homo sapiens)
-SEQ ID NO: 5; Gelsolin (NP_000168.1; corresponding to Homo sapiens)
-SEQ ID NO: 6; Ribonuclease UK114 (P52758.1; corresponding to Homo sapiens)
SEQ ID NO: 7; aminoacylase 1A (NP_000657.1; corresponding to Homo sapiens)
SEQ ID NO: 8; α enolase (AAB88178.1; corresponding to Homo sapiens)
-SEQ ID NO: 9; Keratin 5 (NP_000415.2; corresponding to Homo sapiens)
SEQ ID NO: 10 Parvalbumin α (P20472.2; corresponding to Homo sapiens)
-SEQ ID NO: 11; Ribonuclease 4 (AAA96750.1; corresponding to Homo sapiens)
SEQ ID NO: 12; serine protease inhibitor A3L (P05544.3; corresponding to Rattus norvegicus)
-SEQ ID NO: 13; subunit 1 of COP9 (Q13098.4; corresponding to Homo sapiens)
-SEQ ID NO: 14; Ras-related GTP-binding protein A (NP_006561.1; corresponding to Homo sapiens).

  The% identity was established based on determining the% identity of the amino acid sequence of the protein corresponding to Homo sapiens with respect to the amino acid sequence of the protein corresponding to Rattus norvegicus.

  As understood in the present invention, the term “% identity” refers to the number of amino acid positions over the entire sequence length compared, wherein all of the amino acids at that position are identical.

More preferably, in step b of the present invention at least one protein or any combination thereof is detected and / or quantified,
A protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or any fragment thereof;
• a protein having at least 80%, 85%, 90%, or 95% identity to the amino acid sequence of SEQ ID NOs: 3-11, or any fragment thereof; or • at least 90% of the amino acid sequence of SEQ ID NOs: 12-14, or A protein with 95% identity, or any fragment thereof.

  In the rest of this specification, when reference is made to any of the proteins of the invention, it should be considered that any protein having the above-mentioned% identity can also be detected. Thus, in the rest of this specification, any of the nail mark proteins may be referred to as “protein of the present invention” or “protein of the present invention”. .

  In order to detect and / or quantify the protein of the invention, it is sufficient to detect one or more fragments of the protein. This is because the fragment is a component of the amino acid sequence and structure of the protein.

  Step b of the method of the invention relates to the detection and quantification of the protein or any fragment thereof, and to the detection or quantification thereof. The detection and / or quantification may be performed by any technique known in the art.

  At the same time, as already mentioned above, the present invention can be carried out via any combination of any of the above listed proteins or fragments of the above proteins. Furthermore, the term “any combination thereof” also refers to the fact that one or more of the proteins of the invention may be detected in combination with the detection of any other protein that is different from any of the proteins of the invention.

  Any of the proteins of the present invention is an expression product of a nucleotide sequence. This nucleotide sequence can be, by way of non-limiting illustration, any RNA (eg, mRNA) or any fragment thereof. The nucleotide sequence can also be complementary DNA (cDNA) or any fragment thereof. cDNA is complementary to mRNA and is also a nucleotide sequence that contains exons of genomic nucleotide sequences and no introns. That is, cDNA is a coding sequence. A transcript of the genomic nucleotide sequence of the gene encoding the protein and a transcript of the cDNA of the protein encode the same mRNA and thus the same protein. In the present invention, any RNA or any DNA or any fragment thereof can also be detected instead of or simultaneously with the detection of protein.

  A preferred embodiment relates to a method for providing data useful for determining renal damage, wherein at least one protein selected from the list comprising SEQ ID NOs: 1-14 or any combination thereof is detected and / or Quantified.

  Another preferred embodiment relates to a method for providing data useful for determining renal injury, wherein a protein having at least 60% identity to SEQ ID NO: 1 and / or at least 60% identity to SEQ ID NO: 2. The protein or any fragment thereof is detected and / or quantified. According to a more preferred embodiment, the protein of SEQ ID NO: 1 and / or the protein of SEQ ID NO: 2 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the present invention refers to a method for providing data useful for determining renal damage, wherein a protein having at least 60% identity to SEQ ID NO: 1 or any fragment thereof is detected and / or Or quantified. According to a more preferred embodiment, the protein of SEQ ID NO: 1 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the present invention refers to a method for providing data useful for determining renal damage, wherein a protein having at least 80% identity to SEQ ID NO: 5 or any fragment thereof is detected and / or Or quantified. According to a more preferred embodiment, the protein of SEQ ID NO: 5 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the present invention refers to a method for providing data useful for determining renal injury, wherein the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 5 or any fragment thereof are detected and / or Or quantified.

  Another preferred embodiment relates to a method for providing data useful for determining renal injury, wherein the data obtained in step b is obtained from a control sample in order to find any significant difference. And a step of comparing.

  As understood in the present invention, the term “control sample” refers, by way of non-limiting example, to a sample obtained from an individual who is not suffering from kidney injury (a healthy individual). This type of control sample is a negative control sample or negative control for kidney injury.

As understood in the present invention, the term “significantly significant difference” refers to the presence of the protein of the present invention in an isolated sample or compared to a sample isolated from a healthy individual. It refers to the higher concentration of the protein of the invention in the isolated sample.
Healthy individuals are selected by measuring at least one or more levels of common markers of kidney damage. The common marker is selected from a list including but not limited to creatinine, blood urine nitrogen (BUN) or urine protein.

  A biological sample isolated from an organism (eg, but not limited to a human or other animal) can be a bodily fluid or any cellular tissue from the organism.

  Another preferred embodiment of the invention relates to a method for determining kidney damage, the method comprising the steps of a method for obtaining data as described above, wherein the significant difference is the development of kidney damage in an individual. It further includes being derived from. Thus, this preferred embodiment is a method for diagnosing renal injury.

  Another preferred embodiment relates to a method for providing data useful for determining kidney damage, wherein the kidney damage is acute renal failure. As understood in the present invention, the term “acute renal failure” refers to acute renal failure at any stage (severity) of renal dysfunction. The origin of acute renal failure includes, but is not limited to, genetic, immune system, ischemia, or treatment with any drug. Acute renal failure can be generated by non-limiting exemplary kidney, prostate, bladder, ureter or urethral surgical procedures. As understood in the present invention, the terms “acute kidney injury” and “renal injury” refer to any intensity of acute kidney injury, which may or may not lead to acute renal failure. .

  In another preferred embodiment of the method for providing data useful for determining renal injury or acute renal failure, the biological sample of step a is a body fluid. Body fluids can include fluids that are drained or secreted from the animal's body. Body fluids include, but are not limited to, amniotic fluid surrounding the fetus, aqueous humor, blood, serum, wet / dry fluid, lymph, milk, mucus (including nasal discharge, sputum), saliva, sebum, serum, sweat, tears or Selected from a list containing urine. In a more preferred embodiment, the body fluid is serum. The proteins of the present invention can be found in any biological component present in the body fluids described above, including but not limited to cells or vesicles. In a more preferred embodiment, the body fluid is urine.

  Another preferred embodiment relates to a method for providing data useful for determining renal injury or acute renal failure, wherein the protein is detected and / or detected by electrophoresis, immunoassay, chromatography and / or microarray technology. Or quantified.

  Detection and / or quantification of the proteins of the invention can be performed by any of the techniques described above, or any combination thereof. A protein can be detected by assessing its presence or absence. Detection can be done by specific recognition of any fragment of the protein by any probe and / or any antibody. The protein of the invention or any fragment thereof can be quantified using data that serves as a reference for comparison with data obtained from a control sample and as a reference for finding any significant differences. This significant difference can be helpful in diagnosing kidney damage in the individual who provided the sample in question. In a preferred embodiment, the protein of the invention or any fragment thereof can be detected and / or quantified by electrophoresis and / or immunoassay.

  Electrophoresis is a separation analysis technique based on the movement or migration of macromolecules dissolved in a medium (electrophoretic buffer) through a matrix or solid support as a result of the action of an electric field. The behavior of a molecule depends on its electrophoretic mobility, which depends on load, size and shape. There are various variations of this technique based on the equipment, support and conditions used to perform the protein separation. Electrophoresis includes, but is not limited to, capillary electrophoresis, on-paper electrophoresis, electrophoresis in agarose, electrophoresis in polyacrylamide gels, isoelectric focusing or two-dimensional electrophoresis More selected.

  An immunoassay is a biological test that measures the concentration of a substance in a biological fluid and uses the response of an antibody to any of its antigens. This assay is done using the specificity of the antibody for its antigen. The amount of antibody or antigen can be detected by methods known in the art. One of the most common methods is based on antigen or antibody marking. This marking can be done by non-limiting exemplary enzymes, radioisotopes (radioimmunoassay), magnetic labels (magnetic immunoassay) or immunofluorescence, and can also be performed by aggregation, nephelometry, turbidimetry It can be done by turbidimetry or other techniques including Western blotting. Heterogeneous immunoassays may or may not be competitive. If the immunoassay is competitive, the reaction is inversely proportional to the concentration of antigen in the sample. If the immunoassay is noncompetitive, the result is directly proportional to the concentration of antigen. An immunoassay technique that can be used in the present invention is an ELISA assay (enzyme immunoassay).

  Using chromatographic techniques, molecules can be separated according to, but not limited to, load, size, molecular weight, polarity or redox capacity. The chromatographic technique is selected from, but not limited to, liquid chromatography (fractional chromatography, absorption chromatography, exclusion chromatography, or ion exchange chromatography), gas chromatography or supercritical fluid chromatography.

  The microarray technology of the present invention is based on, for example, immobilizing a molecule that recognizes the protein of the present invention on a solid support. Antibody-based microarrays are the most common type of protein microarray. In this case, the antibody is immobilized on a solid support (the term chip can also be used with reference to a microarray). This antibody is used to capture molecules that allow detection of related proteins, including but not limited to biological samples, cell lysates, serum or urine. As used herein, the term “solid support” refers to a wide variety of materials such as ion exchange resins, adsorption resins, glass, plastics, latex, nylon, gels, cellulose esters, paramagnetic spheres or these. Any combination of these may be mentioned, but the invention is not limited to these.

  According to another preferred embodiment of the method for providing data useful for determining renal injury or renal failure, renal injury or acute renal failure is produced by administration of or exposure of an individual to at least one nephrotoxic factor. Is done. A more preferred embodiment relates to a method wherein the nephrotoxic factor is an aminoglycoside antibiotic.

  Nephrotoxic factors can cause one or more renal pathologies due to administration or exposure. This administration or exposure may occur over a period of time, be limited to a single event, or may be attributed to one or more compounds. The environment of exposure can be the result of unconsciousness, accidental, deliberate, overdose, or the need for treatment (administration). The kidney is an important drainage organ. This is because the kidneys maintain the homeostasis of water-soluble molecules and can actively concentrate certain substances. In general, proximal tubules, distal tubules or tubules can be repaired, but the glomeruli and medulla have a very low tendency to repair.

  The nephrotoxic factor, when administered, may be a pharmaceutical composition (therapeutic agent), a halogen anesthetic, or a compound contained in a functional food or vitamin supplement or nutritional supplement, It is not limited to. A nephrotoxic factor, when exposed, can be, for example, but not limited to, a heavy metal, a plaguicide, or an antimicrobial agent. Antibiotic agents include, but are not limited to, fungicides, herbicides, insecticides, algaecides, molluscicides, tick control agents or rodenticides. Antimicrobial agents include, but are not limited to, bactericides, antibiotics, antimicrobial agents, antiviral agents, antifungal agents, antiprotozoal agents or antiparasitic agents.

  Aminoglycoside antibiotics (aminoglycosides) act by binding bacterial ribosomes to subunits 30S and 50S, inhibit peptidyl tRNA migration, cause misreading of mRNA, and bacteria synthesize viral proteins for their growth Leave it impossible to do. The aminoglycoside antibiotic can be amikacin, arbekacin, gentamicin, ketamicin, neomycin, netilmicin, paromomycin, rhodostreptomycin, streptomycin, tobramycin, apramycin, spectinomycin, hygromycin B, berdamycin, astromycin or puromycin However, it is not limited to these. According to an even more preferred embodiment, the aminoglycoside antibiotic is gentamicin.

  Gentamicin is a broad spectrum antibiotic that is commonly used to treat infections of gram-negative aerobic bacteria. Aminoglycosides are hardly absorbed when administered orally but are rapidly concentrated by the kidneys. In addition, aminoglycosides spread into bacterial cells via channels located in the outer membrane and migrate to the cytoplasm. As a result, gentamicin acts by inhibiting the synthesis of bacterial proteins, but can cause kidney damage to the proximal tubule (especially segment S1 or S2), which can induce acute kidney injury, acute kidney failure Can be caused. In addition, acute renal failure can result from simultaneous or sequential administration of a second nephrotoxic factor. This second compound can be, for example, but not limited to, uranyl nitrate or cisplatin. Uranyl nitrate is a nephrotoxic factor that causes severe renal dysfunction and acute tubular necrosis. Other target organs include the liver, lungs or brain. Cisplatin is an antitumor drug with a broad spectrum and is commonly used to treat testicular, ovarian, bladder, skin, cervical or lung tumors. Cisplatin spreads within cells and functions by entering inside or between DNA strands, causing cell death.

  Another embodiment of the present invention relates to a method for providing data useful in determining renal injury or acute renal failure, wherein the protein is detected from 12 hours after initiation of nephrotoxic factor administration or exposure. Is done. According to a more preferred embodiment, the protein is detected 24 hours after the start of nephrotoxic factor administration or exposure.

Another embodiment of the present invention is to determine renal damage or renal failure caused by gentamycin, to determine renal damage or acute renal failure, to distinguish renal damage or renal failure caused by cisplatin. With respect to methods that provide useful data, the method is performed by detecting and / or quantifying SEQ ID NO: 1 and / or SEQ ID NO: 5 or any fragment thereof. Another more preferred embodiment of the invention relates to a method for differentiating or distinguishing renal damage or renal failure caused by gentamicin from that caused by cisplatin, wherein:
a. When SEQ ID NO: 1 and / or SEQ ID NO: 5 is detected or quantified, the cause of kidney damage or renal failure is administration or exposure of gentamicin;
b. If SEQ ID NO: 1 and / or SEQ ID NO: 5 are not detected or quantified, the cause of renal injury or renal failure is cisplatin administration or exposure.

Thus, the methods of the present invention are useful for differentially determining the cause of renal injury or renal failure. The method can distinguish whether renal injury or renal failure is due to gentamicin administration or exposure or cisplatin administration or exposure. In this case, there are several possibilities:
(I) When SEQ ID NO: 1 and SEQ ID NO: 5 are detected or quantified, or when SEQ ID NO: 1 is detected or quantified, or when SEQ ID NO: 5 is detected or quantified, the cause of kidney damage or renal failure is Gentamicin administration or exposure;
(Ii) When SEQ ID NO: 1 and SEQ ID NO: 5 are not detected or quantified, or when SEQ ID NO: 1 is not detected or quantified, or when SEQ ID NO: 5 is not detected or quantified, renal damage or renal failure is caused by administration of cisplatin. Or exposure.

Another aspect of the invention is a method for predicting the progression of renal damage resulting from administration of at least one nephrotoxic factor, the method comprising:
(A) determining a first concentration of the protein or any fragment thereof in a body fluid isolated from an exposed or unexposed individual of a nephrotoxic factor comprising: Is selected from a list containing:
A protein having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or any fragment thereof;
A protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 3-11, or any fragment thereof; or a protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 12-14, or any thereof Fragment. Second, after the first concentration of the protein in the exposed individual has been determined, or after administration of or exposure to the nephrotoxic factor in an unexposed individual has begun, the concentration in step a is A second concentration of the determined protein in the body fluid of the individual is determined. And
(C) Comparing the second concentration obtained in step b with the first concentration included in step a to find any significant difference. That is, if the first concentration is determined from a sample of an individual exposed to the nephrotoxic factor, the second concentration is measured after the first concentration is determined. Where the first concentration is determined from a sample of an individual that has not been exposed to the nephrotoxic factor, the second concentration is measured after administration of the nephrotoxic factor to the individual or exposure of the individual to the factor has begun. The The second concentration is compared with the first concentration to determine any significant difference. If the second concentration is compared to the first concentration, or if any significant difference is compared to the predetermined concentration, the significant difference can be a higher or lower value.

  In the present invention, the term “predicting progression” can summarize monitoring or managing the progression of renal injury, in other words, distinguishing progression of renal injury resulting from administration of at least one nephrotoxic factor. Say.

  A preferred embodiment relates to a method for predicting the progression of renal injury resulting from administration of at least one nephrotoxic factor, wherein a list comprising SEQ ID NO: 1-14 or any fragment thereof in steps a and b At least one of the more selected proteins, or any combination thereof, is detected and / or quantified.

  Another preferred embodiment relates to a method for predicting progression of renal damage resulting from administration of at least one nephrotoxic factor, wherein a protein having at least 60% identity to SEQ ID NO: 1, and / or The concentration of a protein having at least 60% identity to SEQ ID NO: 2, or any fragment thereof, is determined. According to a more preferred embodiment, the protein of SEQ ID NO: 1 and / or the protein of SEQ ID NO: 2 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the invention relates to a method for predicting the progression of renal damage resulting from administration of at least one nephrotoxic factor, wherein the protein is at least 60% identical to SEQ ID NO: 1 or any of its proteins Fragments are determined. According to a more preferred embodiment, the protein of SEQ ID NO: 1 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the invention relates to a method for predicting the progression of renal damage resulting from administration of at least one nephrotoxic factor, wherein the protein is at least 80% identical to SEQ ID NO: 5 or any of its The concentration of the fragment is determined. According to a more preferred embodiment, the protein of SEQ ID NO: 5 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment of the present invention relates to a method for predicting the progression of renal damage resulting from administration of at least one nephrotoxic factor, wherein the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 5, or these Any fragment of is detected and / or quantified.

  Another preferred embodiment relates to a method for predicting the progression of renal injury resulting from administration of at least one nephrotoxic factor, wherein the renal injury is acute renal failure.

  According to another preferred embodiment of the method for predicting progression of renal injury or acute renal failure, the nephrotoxic factor is an aminoglycoside antibiotic. A more preferred embodiment relates to a method wherein the aminoglycoside antibiotic is gentamicin.

  According to another preferred embodiment of the method for predicting progression of renal injury or acute renal failure, the nephrotoxic factor is cisplatin.

  According to another preferred embodiment of the method for predicting progression of renal injury or acute renal failure, the body fluid is urine or serum.

  Refers to any of the methods of the present invention for obtaining data useful for determining renal injury or acute renal failure, or any of the methods for predicting progression of renal injury as described above or acute renal failure as described above. To that end, the term “method of the invention” can be used.

  According to a preferred embodiment of the method of the invention, the individual is a human. The individual of the method of the present invention may be an animal. This is because the method in question is also useful for veterinary medicine for whistle.

Another aspect of the invention is the use of at least one protein or any combination thereof, said protein as a biomarker for determining renal damage or for predicting progression of renal damage A protein having at least 60% identity to one or two amino acid sequences, or any fragment thereof;
A protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 3-11, or any fragment thereof; or a protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 12-14, or any thereof Selected from the list containing the fragments.

  A preferred embodiment relates to use, wherein a protein selected from the list comprising SEQ ID NOs: 1-14 or any fragment thereof is detected and / or quantified.

  Another preferred embodiment relates to the use wherein the concentration of a protein having at least 60% identity to SEQ ID NO: 1 and / or a protein having at least 60% identity to SEQ ID NO: 2, or any fragment thereof is determined. Is done. According to a more preferred embodiment, the protein of SEQ ID NO: 1 and / or the protein of SEQ ID NO: 2 or any fragment thereof is determined and / or quantified.

  Another preferred embodiment is the use, wherein the protein is selected from a list comprising SEQ ID NOs 1-14 or is a fragment thereof.

  According to another preferred embodiment of the use of the invention, the protein is selected from proteins having at least 60% identity to SEQ ID NO: 1 or fragments thereof. Preferably, the protein is the protein of SEQ ID NO: 1 or a fragment thereof.

  Another preferred embodiment of the invention relates to the use wherein the protein is at least 80% identical to SEQ ID NO: 5 as a biomarker to determine the risk of development of renal injury or as a predictor of progression of renal injury. It is selected from a protein having a sex or a fragment thereof. Preferably SEQ ID NO: 5 or a fragment thereof is selected.

  Another preferred embodiment of the invention relates to the use, wherein the renal injury is acute renal failure.

  This biomarker indicates a change in expression or status of a protein associated with renal injury or progression of renal injury. Once a biomarker is identified, it can be used to diagnose kidney damage or progression of kidney damage or to adapt an individual's treatment (eg, treatment with various drugs or dosing regimens) to such kidney damage Let

  If the treatment alters the presence or concentration of a detected biomarker that is directly related to the risk of developing kidney damage, the biological indicator may be any treatment or exposure to any nephrotoxic agent It serves as an indication of modifying.

  A preferred embodiment of the use of the protein or any fragment thereof is the use when renal damage or progression of renal damage results from administration of at least one nephrotoxic factor. According to a more preferred embodiment of the use of the protein or any fragment thereof, the nephrotoxic factor is an aminoglycoside antibiotic. A more preferred embodiment with another dish is the use when the aminoglycoside antibiotic is gentamicin.

  According to a more preferred embodiment of the use of the protein or any fragment thereof, the nephrotoxic factor is cisplatin.

Another aspect of the invention relates to a kit comprising at least one probe capable of recognizing at least one protein or any combination thereof, wherein the protein comprises:
A protein having at least 60% identity to the amino acid sequence of SEQ ID NO: 1 or 2, or any fragment thereof;
A protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 3-11, or any fragment thereof; or a protein having at least 90% identity to the amino acid sequence of SEQ ID NO: 12-14, or any thereof Selected from the list containing the fragments.

  A preferred embodiment relates to a kit where the probe recognizes at least one protein selected from the list comprising SEQ ID NOs: 1-14 or any fragment thereof or any combination thereof.

  Another preferred embodiment relates to a kit, wherein the probe comprises a protein having at least 60% identity to SEQ ID NO: 1 and / or a protein having at least 60% identity to SEQ ID NO: 2, or any fragment thereof Recognize concentration. According to a more preferred embodiment, the probe recognizes the protein of SEQ ID NO: 1 and / or the protein of SEQ ID NO: 2 or any fragment thereof.

  Another preferred embodiment of the invention relates to the kit, wherein the probe recognizes a protein or fragment thereof having at least 60% identity to SEQ ID NO: 1. Preferably, the probe recognizes the protein of SEQ ID NO: 1 or a fragment thereof.

  Another preferred embodiment of the invention relates to the kit, wherein the probe recognizes a protein or fragment thereof having at least 80% identity to SEQ ID NO: 5. Preferably, the probe recognizes the protein of SEQ ID NO: 5 or a fragment thereof.

  Another preferred embodiment of the invention relates to a kit, wherein the probe is linked to a solid support.

  A probe is generally (although not necessary) a marked material and is used to detect, identify and / or quantify any protein of the invention or fragment thereof. The probe may be, but is not limited to, a probe that reacts with a thiol group, avidin, streptavidin or peptin group. The solid support is preferably a gel (non-limiting exemplary agarose gel or acrylamide gel).

  A preferred embodiment relates to a kit, wherein the probe is an antibody that recognizes the protein or any fragment thereof. The antibody may be polyclonal or monoclonal.

  Another embodiment of the invention is the use of the kit described above in an isolated sample from an individual to determine renal damage or to predict progression of renal damage.

  Another aspect of the present invention is a kit as described above in an isolated sample from an individual for determining the risk of developing kidney damage, for determining kidney damage, or for predicting progression of kidney damage. Is the use of.

  Throughout this specification and the claims, the term “comprising” and variations thereof are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, benefits and features of the present invention are inferred in part from the description herein and in part from the practice of the invention. The accompanying drawings and the following examples are provided for purposes of illustration and are not intended to limit the invention to that form.

[Brief description of the drawings]
FIG. 1 shows the survival rate and the appearance of renal injury markers in rats treated with gentamicin. A: Survival indicates the percentage of animals surviving in each group after 7 days of treatment. B: Western blot analysis of expression levels of kidney injury 1 molecule (KIM-1) and bone morphogenetic protein 7 (BMP-7) in control and kidney homogenates from 3 gentamicin rats after 7 days of treatment A representative image.

  FIG. 2 shows a representative image of the cortex and nipple of a kidney tissue section from a rat treated with gentamicin. Representative of cortical (AD) and papilla (E-H) from renal tissue sections from randomly selected hematoxylin and eosin stained control rats (n = 3) and gentamicin treated rats (n = 3) Image. Images A, B, E, and F were taken at 400 × magnification, and images C, D, G, and H were taken at 1000 × magnification.

  FIG. 3 shows SDS-PAGE separation of urine protein from control rats and rats treated with gentamicin. Proteins were separated by SDS-PAGE on polyacrylamide gels (6% (A) and 15% (B)) and then stained with Coomassie brilliant blue. Proteins present in band (1-24) were determined by LC-ESI-Q-TOF and listed in Table 3. Lane 1: molecular weight pattern, lane 2: control urine, lane 3: gentamicin group urine.

  FIG. 4 shows a two-dimensional (2D) electrophoresis image of a differentially expressed protein. Quantitative analysis of intensity using Image Master 2D Platinum software (GE Healthcare) showed 129 proteins that were differentially expressed between normal and gentamicin urine samples. Spots were marked with numbers corresponding to Table 4 and identified by LC-ESI-Q-TOF and MALDI-TOF. All the gels shown in the figure are 8 representative gels obtained using urine from animals randomly selected from each group. Each group was analyzed in duplicate.

  FIG. 5 shows urinary proteomics by 2D gel images of differentially expressed regIII (A) and gelsolin (B). Spots labeled with numbers corresponding to the table were subjected to quantitative analysis of intensity and identified by LC-ESI-Q-TOF mass spectrometer. Each gel shown in the figure is a representative 8 gels obtained using urine from 4 animals randomly selected in each group. Each group was analyzed in duplicate. The regIIIb protein is spot 1 and has an accession number of P25031, MW 20 kDa, PI 7.56. Gelsolin has spots 2, 3 and 4, and accession numbers are Q68FP1, MW 86.1 kDa, and PI 5.75.

  FIG. 6 shows Western blot analysis of regIIIb (A) and gelsolin (B). A: Western blotting of urine from 6 randomly selected rats treated with vehicle (control), gentamicin or cisplatin. Arrows indicate full length and t-gelsolin fragment. B: Rat serum creatinine and BUN concentrations shown in A. *, P <0.05 vs control; ●, p <0.05 vs gentamicin.

FIG. 7 shows the changes over time of regIIIb (A) and gelsolin (B) in urine. Representative images of western blotting analysis of regIIIb (A) and gelsolin (B) in urine, and NAG excretion and urinary protein of rats treated with gentamicin; serum creatinine concentration; KIM-1, NGAL and LAI-1 Western blotting analysis; representative images of kidney sections stained with hematoxylin plus eosin from rats treated with gentamicin for 3 days. N = 3 in all experiments. ^* , P <0.05 relative to time 0.

  FIG. 8 shows Western blotting analysis, renal perfusion experiments and RT-PCR analysis of serum levels of regIIIb (A) and gelsolin (B). Representative images of western blotting analysis of regIIIb (A.1) and gelsolin (B.1) from randomly selected rats treated with vehicle (control) or gentamicin for 6 days. Renal perfusion experiment: Representative image of western blotting analysis of urinary levels of regIIIb (A.2) and gelsolin (B.2) from rats treated with gentamicin for 6 days. It was then subjected to renal perfusion with Krebs solution. Immediately before the start of perfusion, 1 hour and 2 hours after perfusion; n = 3. A3. RegIIIb (A.3), gelsolin (B.3) and GAPDH of renal tissue by RT-PCR from 3 randomly selected rats treated with vehicle (control) or gentamicin for 3 or 6 days Gene expression.

〔Example〕
[Example 1. Experimental materials and experimental procedures)
1.1 Animals and Experimental Procedures Female Wistar rats with a weight of 200-250 g were used. Animals were placed in individual metabolic cages under controlled environmental conditions for each urine sample collection every 24 hours. Normal food and water were administered as appropriate. Rats were randomly divided into two groups: (i) control group (C), receiving daily placebo ip for 6 days, and (ii) gentamicin group (G), 6 days gentamicin ip ( 150 mg / kg body weight). On day 7, the animals were anesthetized with sodium pentobarbital and the kidneys were perfused by dissection with saline (0.9% NaCl). The kidney was immediately dissected. One was frozen in liquid nitrogen and subsequently kept at −80 ° C. for western blot studies and the other was soaked in 3.7% p-formaldehyde for histological studies. . Blood samples were also obtained in heparinized capillaries at different time points by small incisions at the tail tip. The blood was centrifuged and the serum was kept at −80 ° C. until use. Urine was removed by centrifugation at 10,000 g and 4 ° C. for 10 minutes and kept at −80 ° C. until use.

1.2 Histological studies The kidneys were kept overnight in p-formaldehyde at 4 ° C. Paraffin fixation was then performed and 5 μm tissue sections were cut and stained with hematoxylin and eosin. The photo was taken under an Olympus BX51 microscope connected to an Olympus DP70 color digital camera.

1.3 Biochemical measurements Serum and urine creatinine and urea nitrogen concentrations were measured using commercially available test strips (Roche Diagnostics) and Reflotron® automated analyzers (Roche Diagnostics). The creatinine concentration measurement had a lower detection limit of 0.5 mg / dL. Urine protein concentration was measured by the Bradford method (Bradford, 1976. Anal Biochem, 72: 248-254). Urinary NAG content was indirectly measured by the level of NAG activity. NAG enzymatic activity can be achieved by replacing 3-cresolsulfonephthaleinyl-N-acetyl-D-glucosamine with purple 3-cresol-cresolsulfonphthaleinyl at 580 nm using a plate photometer (Thermo). Measured by a based colorimetric method. To measure NAG activity, a commercially available kit was used according to the manufacturer's instructions (Roche Diagnostics).

1.4 Western Blot Western blots consisted of (i) urine samples (21 μl per sample), (ii) homogenization buffer (140 mM NaCl, 20 mM Tris-HCl pH = 7.5, 0.5 M ethylenediaminetetraacetic acid-EDTA- Tissue agitator (Ultra-Turrax) at 4 ° C. in 10% glycerol, 1% Igepal CA-630, 1 mg / mL aporotinine, 1 mg / mL leupeptin, 1 mg / mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride-PMSF-) Performed with tissue extract (100 μg total protein per sample) prepared by homogenizing the kidney using T8, IKA®-Werwe) or (iii) albumin-free blood serum It was. Albumin was removed from the serum using a column-based, commercially available kit based on immunological retention of rat albumin (Qproteome Murine Albumin Depletion Kit, Quiagen). Samples were separated by electrophoresis in 10-15% acrylamide gels. Immediately, the protein was electrically transferred to an Immobilon-P membrane (Millipore). The membranes are KIM-1 (R & D Systems) bone morphogenic protein 7 (BMP-7, Santa Cruz Biotechnology), NGAL (MBL), PAI-1 (BD Biosciences), Leg IIIb (R & D Systems), and gelsolin (Santa Cruz Biotechnology). ).

1.5 Separation of 1D and 2D (1D and 2D) electrophoretic proteins Urine was concentrated and desalted by forced filtration by centrifugation in a 5K blocked Amicon Ultra column. Protein concentration was measured by the Bradford method. 100 mg total protein for 1D electrophoretic separation was made to run on 6 and 15% acrylamide gels (Mini Protean II system, BioRad, Madrid, Spain), using Coomassie Brilliant Blue G-250 And stained. For two-dimensional electrophoresis (2D), urine proteins were precipitated using a removal kit (GE Healthcare) according to the manufacturer's instructions. 100 mg protein in each sample is 7M urea, 2M thiourea, 4% chaps, 0.5% ampholyte, pH 4-7 or 4.5-5.5, 50 mM dithiothreitol (DTT) and bromophenol blue Rehydrated in, using IPGphor apparatus (GE Healthcare), 18 cm long immobilized pH gradient (IPG) test paper, pH 4-7 or 4.5-5.5 (GE Healthcare, Madrid, Spain) etc. Electrophoresis (500-8,000V) was performed. IPG test strips contain 1% (w / v) DTT, equilibration buffer [50 mM Tis-HCl, pH = 8.8, 6 M urea, 30% (v / v) glycerol, 2% ( w / v) sodium dodecyl sulfate (SDS), 0.01% (w / v) bromophenol blue, pre-equilibrated for 15 minutes and further 2.5% (w / v) iodoacetamide for 15 minutes Pre-equilibrated in the equilibration buffer. The IPG test paper was then transferred to an 18 cm long 12% acrylamide gel and separated by electrophoresis using a SE 600 Ruby instrument (GE Healthcare). The gel was fixed overnight in 30% ethanol and 10% acetic acid and silver stained using a commercial kit (GE Healthcare). Unless otherwise indicated, all reagents are from Sigma.

  For visualization and analysis, stained gels are scanned (Image Sanner, GE Healthcare, Madrid, Spain) and processed by Image Master 2D Platinum 6.0 software (GE Healthcare, Madrid, Spain) for statistical analysis It was done. Spot identification was performed according to the following parameters: (i) Smooth coefficient: 2; (ii) Minimum area: 5 pixels; (iii) Saliency: 100. Analysis was collected visually for artifact elimination. A background was drawn for each individual spot and the individual intensity quantities were normalized by the overall intensity quantity (all spot intensities). As a comparison of the same spots between gels, a minimum 2-fold difference was established to allow for unique expression.

1.6 Two-dimensional liquid chromatography (Chromatofocus + Reverse phase) (2D-LC)
ProteomeLab PF2D system (Beckman Coulter) was used. Concentrated and desalted urine is diluted with optimization buffer (7.5 M urea, 2.5 M thiourea, 12.5% glycerol, 62.5 mM Tris-HCl, pH = 7.8-8. 30 minutes at room temperature). 2, 2.5% (w / v) n-octyl glucoside, 6.25 mM Tris (carboxyethyl) phosphine hydrogen chloride). This buffer was subsequently replaced by starting buffer (pH 8.5 ± 0.1, Beckman Coulter) using a PD-10 column (GE Healthcare). 3.5 ml of the first fraction was collected and the protein concentration was quantified. Next, 1 mg of each sample was placed on a chromatofocus ion exchange column and eluted into pH 0.3 fractions based on the isoelectric point by means of a linear descending pH gradient (8.5-4). . Each pH fraction was subsequently run in a linear gradient of 5-100% with 0.08% TFA in acetonitrile at 0.75 mL / min and A above is 0.1% TFA in water. Separation at 50 ° C. based on protein hydrophobicity by reverse phase high performance liquid chromatography using a C18 non-porous column (RP-HPLC). A 0.6 min reverse phase fraction was collected and held at -80 ° C. Absorbance was measured at 214 nm in two dimensions and the data was analyzed using 32 Karat and Delta View software (Beckman Coulter).

1.7 Identification of proteins from 1D, 2D, and 2D-LC separations Bands and spots of interest from 1D and 2D separations (respectively) were excised from the gel. Each gel cut was dehydrated in acetonitrile, dried in vacuo, and the residue was resuspended in NH ₄ HCO ₃ . Therefore, samples from 1D, 2D, and 2D-LC were processed identically. Samples were subsequently reduced with 10 mM DTT in 50 mM NH ₄ CO ₃ at 56 ° C. Subsequently, the protein was digested in gel with porcine trypsin (Promega) for 30 minutes at 4 ° C. The peptide was subsequently extracted with 0.5% trifluoroacetic acid (TFA). The solution was vacuum dried and the peptide was dissolved in 0.1% formic acid under breakage. The peptide-containing solution was injected into the LC9 ESI-QUAD-TOF mass spectrophotometer QSTAR XL (Applied Biosystems) using 1100 micro HPLC (Agilent). A 150 × 0.32 mm (5 m) pore size Supelco column ((Discovery BIO) was used at a flow rate of 7 L / min. MS / MS spectra were obtained. Protein identification was performed using a non-overlapping protein sequence database ( Swiss Prot and NCBI) using MASCOT software (www.matrixscience.com) Mass tolerance was set at 50 ppm, MS / MS tolerance was 0.5 Da, taxonomic When identified by MASCOT feasibility analysis, only the relevant relevant were considered, and at least one peptide observing an ion score of 20 or higher was set as the tolerance threshold In some cases, the LC-ESIQUAD-TOF procedure does not identify unclear proteins, Alternatively, for confirmation at randomly selected spots, the protein was used with the Ultraflex I MALDI-TOF mass spectrophotometer (Bruker Daltonics) at the Proteomics Service of the University of Salamanca-CSIC Cancer Research Center (Salamanca, Spain) Identified.

1.8 Protein Identification by Mass Spectrometry The spot of interest from the 2D separation was excised from the gel, dehydrated with acetonitrile, vacuum dried and the residue resuspended with NH ₄ HCO ₃ . The sample was subsequently reduced with 10 mM DTT in 50 mM NH ₄ CO ₃ at 56 ° C. and alkylated with iodoacetamide in 50 mM NH ₄ CO ₃ . The protein is digested in gel with porcine trypsin (Promega), the peptide is extracted with 0.5% trifluoroacetic acid (TFA), vacuum dried, and 0.1% (v / v) formic acid. Redissolved. The peptide-containing solution was injected into the LC9 ESI-QUAD-TOF mass spectrophotometer QSTAR XL (Applied Biosystems) using 1100 micro HPLC (Agilent). A Supelco column (Discovery BIO) with a pore size of 150 × 0.32 mm (5 μm) was used. An MS / MS spectrum was obtained. Protein identification was performed using MASCOT software (www.matrixscience.com) against non-overlapping protein sequence databases (Swiss Prot and NCBI). The mass tolerance was set at 50 ppm, the MS / MS tolerance was 0.5 Da, and the taxonomic condition was Rattus. When identified by MASCOT likelihood analysis, only the relevant ones were considered, and at least one peptide observing an ion score of 20 or higher was set as the tolerance threshold. For confirmation, several proteins were identified using an Ultraflex I MALDI-TOF spectrophotometer (Bruker Daltonics).

1.9 Gene Expression Analysis RT-PCR amplification of Leg IIIb, Gelsolin, and GAPDH were performed using the following primers: SEQ ID NO: 15 and SEQ ID NO: 16 for rat Leg IIIb; SEQ ID NO: for rat gelsolin 17 and SEQ ID NO: 18; for rat glyceraldehyde 3-phosphate dehydrogenase (GADPH), performed with SEQ ID NO: 19 and SEQ ID NO: 20. PCR conditions are: 1 × (95 ° C. × 4 min); 30 × (95 ° C. × 1 min + Tmx 1 min); 1 × (72 ° C. × 10 min); where Tm for Leg IIIb is 55.5 ° C. and 55.0 for gelsolin And 55.9 ° C. relative to GADPH.

1.10 Kidney drainage study At the end of the treatment, with some modifications, as described elsewhere (69), rats treated with gentamicin for 6 days were anesthetized and the kidneys An extracorporeal circuit for perfusion was installed. Briefly, the right renal artery, vein and ureter were combined. The left renal artery, vein and bladder were cannulated. Oxygen was added, warm (37 ° C.), Krebs dextran solution [Krebs solution (118.3 mM NaCl, 4.7 mM KCl, 1.8 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 0.026 mM EDTA, 11 .1 glucose, pH = 7.4), 40 g / L dextran (molecular weight 64K-76K)] was perfused through the renal artery at 3 mL / min and discarded through the renal vein. The urine fraction is placed in the bladder before initiating perfusion with Krebs solution (when blood is still passing through the kidney) and for 2 hours after initiating perfusion with Krebs solution. All urine samples were collected from the prepared catheters and kept at −80 ° C. until analyzed by Western blot for the presence of Leg IIIb and gelsolin.

1.11 Statistical analysis Data are expressed as the mean ± standard error of n experiments, as indicated in each case. Except for proteomics results studies (as indicated above), statistical comparisons were evaluated by one method, ANOVA analysis. Normal distribution was evaluated (at least 3 times, following the following tests): residue normality due to asymmetry, residue normality due to kurtosis, residue normality due to inclusion, and modified Leven dispersion Uniformity. A Scheffe test was used to determine statistical significance. A value of p <0.05 was considered important.

[Example 2. Characteristics of gentamicin-induced kidney damage
After 7 days of treatment, gentamicin contributed to significant kidney damage (acute kidney injury or acute kidney failure) which was about 50% of the associated mortality (FIGS. 1 and 2). Surviving animals followed a small but important course of weight loss and polyuria. Acute renal failure was further characterized by a dramatic increase in serum creatinine and BUN concentrations indicating GFR reduction. NAG excretion also increased extensively, indicating widespread tubular damage. Proteinuria was also evident in the urine of animals treated with gentamicin (Table 2).

[Example 3. (Histological study of the kidney)
A section of kidney stained with hematoxylin-eosin (FIG. 2) shows clear tubular necrosis of gentamicin rats, where extensive epithelial destruction can be observed. The important changes in the glomeruli are not clear. At the dermal papilla level, obstruction of the collection tube by hyaline material is common in animals treated with gentamicin. These studies provide morphological support for at least some of the widespread renal dysfunction observed.

[Example 4. Analysis of peculiar proteomics of urine)
In this study, three different proteomic approaches (1D-SDS-PAGE, 2D, and 2D-LC) and two mass spectrometry techniques (LC-ESI-QUAD-TOF and MALDI-TOF) are used for protein identification. It was. This combination proved to be partially overlapping and partially non-overlapping. A typical result of 1D is shown in the figure. FIG. 3 represents a typical SDS-PAGE profiling of urine from control and gentamicin rats, where 24 specifically abundant bands were extracted and analyzed from the SDS-PAGE. One contained several proteins. Bands 13-16 were most abundant in the control group, while bands 1-12 and 17-24 were most abundant in the gentamicin group. The proteins identified by this technique are listed in Table 5.

  In preliminary studies, the protein distribution profiles of control and gentamicin urine were determined by isoelectric point. Most proteins were contained at a pH between 4-7. In preliminary studies, the authors used IPG test strips covering a wide pH range (2-10). Because of defects in the range above pH 7, and because relatively few proteins were lost in the pH 7-10 range, the authors experimented in the 4-7 pH range for the first dimension of 2D separation. Decided to do. The upper panel of FIG. 4 shows a typical image of a 2D gel (pH range 4-7) of a urine sample from a control rat treated with gentamicin. Very similarities were observed between samples from the same group of animals, and high reproducibility was obtained when 2D separation was repeated with the same sample. Many proteins were concentrated in the pH range of 4.5-5.5. For this reason, the same 2D separation of the same urine sample was also performed in this pH range. A prominent image of this is shown in the lower panel of FIG. In both cases, unique and statistically significant spots were recognized and numbered for chemical identification between the control and gentamicin groups. (FIG. 4 shows the numbers associated with spots for groups where specific proteins appear more abundantly).

  The urinary proteome of both groups is statistically different. Initially, significantly more spots were identified in the urine of gentamicin treated rats than in the urine of control rats. In the pH range of 4-7, 606 spots were identified from the control group and 933 spots from the gentamicin group. In the pH range of 4.5 to 5.5, the number of spots was 358 and 724, respectively. Of these, 129 specifically expressed spots were found between both groups (37 overexpressed spots in the gentamicin group and 92 spots in the control group). . Mass spectrometry data of these 129 spots identified 98 of them in the protein database as corresponding to 34 protein isoforms or post-transcriptionally modified variants (group of gentamicins In the control group increased 11; see Table 4).

  Complementary studies of the specific proteome of urine from two control animals and two gentamicin treated animals were performed according to the 2D-LC procedure. The similarity between samples of the same group was good and the reproducibility of the results of different experiments using the same sample was also very high. MS analysis of specifically abundant fractions identified 22 proteins (see Table 5). Many of these proteins were found according to 2D techniques such as 2-HS-glycoprotein, hemopexin, serum albumin, T1 kininogen, transferrin, vitamin D binding protein, and urine proteins 1, 2, 3 It was consistent with the one. Many of these proteins have also been identified in several reactions confirming the presence of variants or isoforms. Interestingly, this approach identified several proteins that were not detected by 1D and 2D. This is the case for example with 2-microglobulin and cystatin C. One thing to note is that as a result of the liquid fractionation, a better range of sequences was obtained for most of the proteins identified according to the 2D approach.

  The method of protein separation using specifically 1D, 2D and 2D-LC is not only partially overlapping but also non-overlapping and revealing their complementary values Also proved.

[Analysis of the results of Examples 1 to 4]
In the present invention, urine was selected as a sample of individuals suitable for routine analysis at the population scale. In the case of kidney disease, this is not only convenient due to the availability of simple non-traumatic but also pathological activities that make it easy to collect important diagnostic material from the tissue. It is also for direct contact with the tissue that is happening.

  Urine sampling can be performed 24 hours after the start of treatment or administration of aminoglycoside antibodies to random urine samples. The 24 hour sampling allows for more precise and more accurate normalization through the calculation of the daily excretion of any protein of the invention (marker). However, its use in clinical context is limited to a subset of patients. On the other hand, random samples generally provide better ones for population analysis. However, the level of presence of potential markers can strongly depend on urine concentration and can alter results and conclusions. Attempts to standardize urine concentrations (ie, due to urinary creatinine concentrations) can lead to false results because the parameters of normalization often change in pathological conditions.

  One attempt to resolve these inconveniences is as a fraction of all proteins whose presence in the urine is excreted as a result of an attack (eg, a drug), whether it is increased or decreased during the relevant period ) To be done by creating a urine profile of any protein of the invention that varies. For application in clinical practice, the size of the marker protein in a particular diagnosis must be the result of normalization to the total amount of protein in the analyzed urine sample. For this purpose, therefore, in this study, we standardize urine samples by protein content using the same amount of protein per sample, and then identify the urine samples of the present invention identified by specific proteomic techniques. The relative abundance of any protein was investigated.

From a theoretical point of view, the increase or decrease in absolute or relative abundance of any protein of the invention in urine can be the result of several factors: (i) Changes in glomerular filtration And changes in the secretion of protein tubule tissue from the blood or extracellular kidney space; (ii) markers may be from damaged or repairing kidney tissue; (iii) changes The abundance is generally the result of disturbed reabsorption capacity, or can be specifically also at the level of tubule tissue; (iv) Finally, very importantly, the marker is present It may be derived from altered, unaltered synthesis, signaling pathways, transmembrane transport or exocytosis, or from an extracellular compartment in living kidney tissue.

  In the present invention, through the use of proteomics techniques, a number of marker proteins for renal damage appearing increased in the urine of nephrotoxicated rats with gentamicin relative to healthy (control) rats are found. (Table 6). It must be taken into account that the excretion of these marker proteins has been underestimated since 14 rats in the gentamicin group showed overt proteinuria. This means that the daily secretion of marker proteins identified as increasing in these animals is several times higher than their relative abundance in the urine group (in the gentamicin group). Protein excretion is 5 times higher than in the control group, Table 2). Analysis of 1D and 2D electrophoretic profiles of proteins in urine from both groups (FIGS. 3 and 4) showed that the relative abundance of low molecular weight proteins (<35 KDa) was in control rats. It clearly shows that it is expensive. However, medium molecular weight (MWW) (35-65 KDa) proteins are relatively more abundant in gentamicin treated rats. In the latter, it is particularly clear from the 1D experiment that a high molecular weight protein (HMW) (65 KDa), which is not present in the rats from the control group, appears as a result of treatment with gentamicin. These large proteins can be found above approximately 200 KDa (Figure 3, bands 17-22). The presence of HMW protein in the urine is usually considered a symptom of glomerular filtration barrier (GFB) injury, which increases tolerance to larger proteins that are normally excluded from ultrafiltration. However, high molecular weight proteins can appear as a result of exfoliation of the structure of the kidney in direct contact with the urine, such as filtration barriers, different tubular tissue parts, structures forming the ureters or bladder. Nephritis-type proteinuria, which occurs as a result of the apparent dysfunction of the glomerular filtration barrier, is expected to produce the appearance of a non-specific range of molecular weight proteins in the urine. However, it can also be considered that more specific and subtle damage would only contribute to the appearance of some high molecular weight proteins in the urine. In the case of animals treated with gentamicin of the present invention, the upper limit of the size of the protein that is usually filtered (eg, approximately the size of albumin) with most of the proteins associated with some of the proteins above the threshold Appeared concentrated around or smaller.

  However, along with these HMW proteins, the excretion of many low molecular weight (LMW) proteins also increases after treatment with gentamicin (see Table 6).

  These data, as a whole, are highly nephrotoxic and damage the kidneys, despite the fact that this gentamicin regime produces significant renal dysfunction associated with 50% mortality. It shows that only the filtration function of the glomerular filtration barrier was changed gently (FIGS. 1 and 2). ).

  The effects of tubular reabsorption and secretion processes on the final composition of urine are not only the result of the effects of gentamicin on the level of the tubules. Ultrafiltration compositions also alter protein reabsorption specifically, possibly through a competitive process. Thus, the protein content specifically altered in ultrafiltration as a result of this fact, in addition to providing a specific urinary marker of attack, also modifies its own tubule process.

  The results seen in FIGS. 1 and 2 point to apparent tubular dysfunction caused by gentamicin, which significantly reduces the recovery of the filtered protein at the level of the tubule (Table 2).

  Table 6 lists mainly MMW and LMW proteins. As in many other kidney diseases, most of these proteins are blood-derived, and after treatment with gentamicin, their urine output shows an increase, which is a decrease in tubules Prove a general model of reabsorption.

  In common with other kidney diseases, the specifically detected proteins (Table 6) are cathepsin B, several proteolytic enzymes (alpha-1-antitrypsin, cystatin C, inter-alpha-trypsin inhibitor, serpin) A3L and various acute and immune response proteins (alpha-1-microglobulin, alpha-2-HS-glycoprotein, ceruloplasmin, complement C3 and C9, haptoglobin, hemopexin, immunoglobulin, lysozyme C, T kininogen Their increased excretion is combined with the reabsorption of unbalanced tubule tissue and ultrafiltration (due to their high levels in the blood, not just as a result of overfiltration) Can be explained by the increased abundance.

  In all of the specifically detected proteins (Table 6), the following increases in protein urine values have biomarker values along with diagnostic values for kidney damage: protein leg 3B, fetuin B, Ras-related GTP Binding protein A, serpin A3L, COP9 subunit 1, gamma subunit of ATP synthase, gelsolin, ribonuclease UK114, aminoacylase 1A, alpha-enolase, keratin-5, parvalbumin alpha, ribonuclease 4, serpin A3K. To date, these proteins have never been involved in obtaining useful data for diagnosing kidney disease or used in the diagnosis itself. With the exception of gelsolin and serpin A3K, these proteins are usually not present in the blood. The increase in any of the above proteins in the blood is the result of a phenomenon that occurs in the kidney caused by gentamicin. There are also important candidates to further explore their use in the prognosis of kidney damage and / or acute renal failure caused by aminoglycoside antibiotics such as gentamicin.

  Furthermore, analysis of specific proteomics of the renal cortex has shown that gentamicin is (i) gluconeogenesis and glycolysis (eg fructose 1,6-bisphosphatase and alpha-enolase); (ii) fatty acid transport and metabolism (eg (Iii) fatty acid transport protein, acetyl-CoA carboxylase, methylacyl-CoA racemase); (iii) citrate cycle (eg, ATP-specific succinyl CoA synthase, malate dehydrogenase); and (iv) stress response (eg, moesin, SPI1 , DnaK-type molecular chaperone) was revealed to be a factor of protein over-regulation. One exception is alpha-enolase, which is also increased in the urine of test rats treated with gentamicin (Tables 4 and 6). This deviation in the modification of the protein model between kidney tissue and urine is probably due to the fact that most of the biomarker proteins of the invention derived from the kidney are not the result of increased product, but rather (i) kidney Or (ii) tissue and cell debris excreted, peeled off or damaged in the urine.

[Example 5. Analysis of unique proteomics of urine. Leg IIIb and gelsolin. ]
A typical image of a 2D gel (pH range 4-7) of urine from a control and gentamicin treated rat is shown in the upper panel of FIG. Many proteins are concentrated in the range of pH 4.5-5.5. Therefore, 2D separation in this pH range was also performed using the same urine. These latter exemplary images are shown in the lower panel of FIG. Very similarities were observed between samples from the same group of animals, and high reproducibility was obtained when repeated 2D separations using the same sample for quality confirmation. However, the urinary proteome of both groups is statistically different. Statistically significant, unique spots between the control and gentamicin groups were recognized and numbered for chemical identification. Mass spectrometric analysis identified three proteins increased in urine of gentamicin-treated rats that showed potential interest after removing most of the other proteins, usually found in different proteinuria conditions Was revealed. They were identified as regenerating islet-derived protein 3 beta (Leg IIIb) and gelsolin (FIG. 5).

[Example 6. Leg IIIb and gelsolin are specifically excreted in the urine of rats treated with gentamicin. ]
These proteins at increased urine levels in the urine of gentamicin treated rats were confirmed by Western blot analysis. In addition, urine from rats treated with the cisplatin nephrotoxic regime was also analyzed. FIG. 6-B shows serum creatinine concentration and BUN data from 6 control rats, 6 rats treated with gentamicin, and 6 rats treated with cisplatin. The data demonstrates that the treated animals showed obvious kidney damage. Their urine was also analyzed for leg IIIb and gelsolin content. FIG. 6A clearly shows that Reg IIIb in urine levels is significantly increased only in gentamicin-treated animals, despite the same kidney damage in cisplatin-treated rats. Yes. The gelsolin western blot showed two reactive bands. The higher one matches the full-length protein, while the lower one matches the fragment. The presence of gelsolin in the reactive band was further reconfirmed by MS / MS mass spectrometry. Treatment with gentamicin induces the appearance of both full-length gelsolin and a 43 kDa fragment in urine. However, there was no full-length band in urine from all rats treated with cisplatin except one of them. Extensive analysis of urine from other cisplatin-treated rats indicates the absence of full-length bands.

[Example 7. Over time evolution of urinary excretion of leg IIIb and gelsolin We further analyzed the evolution of urinary excretion of these proteins in rats treated with gentamicin over time. FIG. 7 shows the temporal profile of kidney damage caused by gentamicin. Serum creatinine, NAG excretion, proteinuria and demonstrated by the evolution of three sensitive markers of urinary level of kidney damage such as KIM-1, NGAL, and plasminogen activator inhibitor 1 (PAI-1) As shown, significant damage has occurred only after 4 days of treatment. Consistent with the accumulated findings (44), serum creatinine is the least sensitive of all markers tested, and histological analysis of kidney sections after 3 days of treatment showed that tubule damage Indicates that it cannot be seen. At this point, tubulin epithelial cells from gentamicin accumulation in the endosomal compartment (45, 46) and other literature reports reporting changes in the exosecretory pathway and endosomal transport (47, 48). It is remarkable that the cytoplasm of the air is turned into a gun. In this scenario, Western blot analysis showed that leg IIIb appeared in the urine, with the most other markers of kidney damage susceptibility starting on day 4. Interestingly, urinary gelsolin (a fragment of 43 kDa) is already on day 1 well before the emergence of all other markers, including KIM-1, PAI-1, NGAL, and NAG. Appears and keeps a high appearance during processing.

[Example 8. Origin of urine leg IIIb and gelsolin]
Western blot analysis of albumin-deficient sera from control and gentamicin-treated rats showed that Reg IIIb was absent (relative to the detection limit of this technique) in the blood compartment, whereas gelsolin was found . Moreover, gentamicin slightly increases this latter in serum levels (Figure 8B). Gene expression analysis performed in kidney tissue by RT-PCR showed that these two proteins are normally expressed in the kidney. Treatment of rats with gentamicin does not alter the expression pattern of gelsolin kidneys, but is still when no detectable kidney damage has occurred (FIG. 7), as early as day 3 leg IIIb It induces an increase in gene expression (FIG. 8-A) and on day 6 the expression of leg IIIb is highest.

  To find out if these urinary proteins that should flow to the urine through the glomerular filtration barrier are blood, we added gentamicin added to the Krebs solution (which contains dextran to compensate for the inflation pressure) The kidneys of the treated rats were perfused for 6 days. We have found that leg IIIb and gelsolin can still be detected in urine just before replacing renal blood flow with Krebs solution (FIG. 8). However, once the renal blood flow was replaced with Krebs solution flow, gelsolin disappeared from the urine and the upper band of leg IIIb was still detected, while the lower band disappeared.

[Analysis of the results of Examples 5 to 8]
Nephrotoxicity causes considerable health and economic problems worldwide. That is an important reason for the failure associated with the process of drug discovery, leading to the disposal of non-clinically interesting molecules. Most importantly, about 25% of the 100 most used drugs in the intensive care unit are potentially nephrotoxic. Moreover, it is estimated that nephrotoxicity accounts for 10-20% of acute kidney failure. An important aspect of optimal clinical handling of AKI is early diagnosis. Significant progress has been made in the last decade on progressively earlier detection based on new, more sensitive urine markers. (Vaidya VS, Ferguson MA and Bonventre JV, 2008. Annu Rev Pharmacol Toxicol, 48: 463-493). However, AKI diagnosis can still be improved in individual medicines for enhanced theranostics and more personalized medicines. In this paper, we provide some evidence of a novel urinary marker to potentially differentiate gentamicin nephrotoxicity from that caused by cisplatin. They will help clarify the pharmacological profile of gentamicin, as well as improve clinical utility.

  Leg IIIb and full-length gelsolin have the potential for a specific or etiological diagnosis of gentamicin nephrotoxicity. They appear in the urine of rats with overt renal damage induced by gentamicin, but are not present in the urine of rats with a similar degree of kidney damage given by cisplatin. Leg IIIb is a 17 kDa member of the calcium-dependent lectin (C-type lectin) superfamily that contains several secreted protein products of four genes (Reg I, II, III, and IV) (Zhang YW , Ding LS and Lai MD, 2003. World J Gastroenterol, 9: 2635-2641). Leg genes have been found in different mammalian species, including humans, rats, and mice. The rat leg gene is located in the chromosomal region of 4p33-q34. In humans, all leg genes other than leg IV are located in the 2p12 region. In general terms, leg family proteins are most notably involved in tissue regeneration in many biological and pathological situations, including not only pancreatitis, but also liver damage, diabetes and cancer (Zhang YW, Ding LS and Lai MD, 2003. World J Gastroenterol, 9: 2635-2641). Our experiments suggest that leg IIIb may be involved in kidney damage and repair during gentamicin treatment and, importantly, it may be used as a specific urinary marker. Since we could not detect this protein in serum, we thought that urinary leg IIIb could occur in kidney tissue. Indeed, our data show that expression of leg IIIb is strongly induced by gentamicin in the kidney prior to urinary and serum markers of toxicology and histological findings (treatment). Day 3 of FIG. 8-A). The development of Reg IIIb kidneys for treatment with gentamicin is further supported by our experiments in renal perfusion. We observed Leg IIIb in the urine even when the blood flow of the kidneys of rats previously treated with gentamicin was rapidly replaced with perfused Krebs solution (FIG. 8-A.2). . Urine leg IIIb appears as a double band in the Western blot analysis, consistent with the two spots of the 2D gel. However, when blood is replaced by Krebs solution in the gentamicin-treated rat kidney circuit, only the upper band is detected in urine. We can only speculate that the lower band corresponds to a proteolytic fragment produced by serum proteolytic enzymes, or renal proteolytic enzymes activated by serum components.

  Gelsolin is a highly conserved 82 kDa protein of the gelsolin superfamily. Remodeling in many normal cellular processes, including cytoskeletal tissue and motility, signaling, apoptosis (Kwiatkowski DJ, 1999. Curr Opin Cell Biol, 11: 103-108); and like inflammation, cancer, amyloidosis Is involved in various pathophysiological conditions (Spinardi L and Witke W, 2007. Subcell Biochem, 45: 55-69). Gelsolin is expressed and secreted in many cell types and is usually found in vertebrate blood. Gelsolin is known as a substrate for the agonist caspase 3 which yields a 42 kDa proteolytic fragment that is involved in the regulation of efflux and apoptosis (t-gelsolin; Sakurai N and Utsumi T, 2006. J Biol). Chem, 281: 14288-14295). Our results indicate that urinary gelsolin can also be improved as a specific diagnostic marker of gentamicin nephrotoxicity. In fact, a band consistent with the full-length protein in Western blot studies appears in the urine of gentamicin-treated rats but is rarely present in cisplatin-treated rats. In contrast, the ~ 43 kDa band in our gel, such as t-gelsolin, is common to both the gentamicin and cisplatin groups. The results shown in FIG. 8 indicate that gelsolin gene expression is not altered in rat kidneys treated with gentamicin (relative to controls). They further show that gelsolin disappears from the urine when the renal blood flow is replaced by Krebs solution, which is probably filtered from the blood through the glomerular filtration barrier. Suggests that This is why full-length gelsolin is detected in the urine of rats treated with gentamicin, but not in those treated with cisplatin, but t-gelsolin appears after both treatments. I can explain that. Gentamicin alters the function of GFB leading to the promotion of specific protein filtration. The cation charge of gentamicin polycations alters the electrostatic function of GFB, promotes the permeability of negatively charged proteins (such as gelsolin, pI = 5.75) and reduces the sieving coefficient of positively charged ones (Cojocel et al., 1983. Toxicol Appl Pharmacol, 68: 96-109). It has been reported that the screening function of GFB does not change for cisplatin. In this case, the full length gelsolin should be excluded from passing through the GFB due to size limitations. However, t-gelsolin is not supplemented in the blood in any case because it has a lower molecular weight and can be filtered more easily through GFB. In the case of tubular necrosis, a larger amount of protein scapes the reabsorption capacity of the impaired tubule, which can be detected in the urine. As shown in FIG. 7, t-gelsolin is a conventional and novel AKI marker, the latter being gentamicin treated significantly faster than those containing KIM-1, NGAL, NAG and PAL-1. Appear in the urine of rats. This can also be used for early monitoring of gentamicin nephrotoxicity.

  This study presents two novel approaches for the specific diagnosis of gentamicin nephrotoxicity that need to be further improved in preclinical and clinical settings for better theranotic use and efficacy of this drug. Urine biomarker candidates are provided. Moreover, the principles of potential application of the etiological diagnosis of AKI to clinical patients who progress to complex conditions that potentially affect renal integrity, including multidrug administration Insist on proof. The etiological diagnosis should be applied in clinical practice and in many other potentially nephrotoxic drugs that are widely used for AKI's pre-renal and post-renal factors. This outlines the tendency of the markers to specifically differentiate the occurrence of undesirable renal effects in order to appropriately and selectively reshape the clinical treatment and optimal treatment plan of the at-risk patient. Will be able to show.

FIG. 2 is a graph showing survival rate and appearance of kidney damage markers in rats treated with gentamicin. FIG. 5 shows representative images of cortex and nipple of kidney tissue sections from rats treated with gentamicin. FIG. 3 shows SDS-PAGE separation of urine proteins from control rats and rats treated with gentamicin. FIG. 2 shows a two-dimensional (2D) electrophoresis image of a differentially expressed protein. FIG. 5 shows urinary proteomics by 2D gel images of regIII (A) and gelsolin (B) that are differentially expressed. It is a figure which shows the western blotting analysis of regIII (A) and gelsolin (B). It is a figure which shows the time-dependent change of regIIIb (A) and gelsolin (B) in urine. FIG. 6 shows Western blotting analysis, renal perfusion experiment and RT-PCR analysis of serum levels of regIIIb (A) and gelsolin (B).

Claims (52)

A method of providing useful data for determining kidney damage,
In biological samples isolated from individuals,
At least one or any thereof of a protein selected from the list in the amino acid sequence of SEQ ID NO: 1 includes a protein having at least 80% identity with the a protein, or 100% identity to the amino acid sequence of SEQ ID NO: 2 to 14 A protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 as the protein to be detected and / or quantified includes the step of detecting and / or quantifying the combination of ,Method.   The method of claim 1, wherein at least one protein selected from the list comprising SEQ ID NOs: 1-14, or any combination thereof is detected and / or quantified.   2. The method of claim 1, wherein a protein that is at least 80% identical to SEQ ID NO: 1 is detected and / or quantified.   4. The method of claim 3, wherein the protein of SEQ ID NO: 1 is detected and / or quantified. The method of claim 1 , wherein the protein of SEQ ID NO: 5 is detected and / or quantified.   The method of claim 2, wherein the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 5 are detected and / or quantified. The method according to any one of claims 1 to 6 , further comprising the step of comparing the obtained data with at least one control sample in order to find any significant difference. The method of claim 7 , wherein the significant difference comprises from the development of the kidney injury in the individual. The renal injury is acute renal failure, the method according to any one of claims 1-8. 10. The method according to any one of claims 1 to 9 , wherein the renal injury or renal failure results from administration of or exposure to at least one nephrotoxic factor. The method according to claim 10 , wherein the nephrotoxic factor is an aminoglycoside antibiotic. The method according to claim 11 , wherein the aminoglycoside antibiotic is gentamicin. 13. The method according to any one of claims 5 to 12 , wherein the protein is detected 12 hours after administration of the nephrotoxic factor or exposure to the factor. The method according to any one of claims 5 to 12 , wherein the protein is detected after 24 hours. The method according to any one of claims 5 to 10 , wherein the nephrotoxic factor is cisplatin. 16. A method according to any one of claims 3 to 15 , for determining a factor of the renal injury or renal failure or for differentiating renal injury or renal failure caused by gentamicin from renal injury or renal failure caused by cisplatin. The method according to item. The method according to claim 16 , comprising:
a. When SEQ ID NO: 1 and SEQ ID NO: 5 are detected and / or quantified, the cause of renal injury or renal failure is administration of gentamicin or exposure to gentamicin;
b. A method wherein the cause of renal damage or renal failure is administration of cisplatin or exposure to cisplatin when SEQ ID NO: 1 and SEQ ID NO: 5 are not detected and / or quantified. The method according to any one of claims 1 to 17 , wherein the biological sample is a body fluid. The method of claim 18 , wherein the body fluid is urine. The method of claim 18 , wherein the body fluid is serum. 21. A method according to any one of claims 1 to 20 , wherein the protein is detected and / or quantified by electrophoresis, immunoassay, chromatography and / or microarray technology. A method of preventing progression of renal damage resulting from administration of at least one nephrotoxic factor comprising: a. In body fluids isolated from individuals exposed to or not exposed to nephrotoxic factors,
At least one or any thereof of a protein selected from the list in the amino acid sequence depicted in SEQ ID NO: 1 comprising the protein having at least 80% identity with the a protein, or 100% identity to the amino acid sequence SEQ ID NO: 2 to 14 Determining the first concentration of the combination, wherein the protein from which the first concentration is determined includes at least a protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
b. Determining a second concentration of the protein or any combination thereof, wherein the second concentration is determined after determining the first concentration in the body fluid of the exposed individual or the exposed After the administration of or exposure to said nephrotoxic factor in an individual who has not, in step a in body fluids from the individual, and c. Comparing the second concentration obtained in step b with the first concentration obtained in step a to find any significant difference. 23. The method of claim 22 , wherein at least one protein selected from the list comprising SEQ ID NOs: 1-14, or any combination thereof, is detected and / or quantified in steps a and b. 23. The method of claim 22 , wherein a protein that is at least 80% identical to SEQ ID NO: 1 is detected and / or quantified. 25. The method of claim 24 , wherein the protein of SEQ ID NO: 1 is detected and / or quantified. 23. The method of claim 22 , wherein the protein of SEQ ID NO: 5 is detected and / or quantified. 24. The method of claim 23 , wherein the protein of SEQ ID NO: 1 and the protein of SEQ ID NO: 5 are detected and / or quantified. 28. The method according to any one of claims 22 to 27 , wherein the renal injury is acute renal failure. The method according to any one of claims 22 to 28 , wherein the nephrotoxic factor is an aminoglycoside antibiotic. 30. The method of claim 29 , wherein the aminoglycoside antibiotic is gentamicin. The method according to any one of claims 22 to 28 , wherein the nephrotoxic factor is cisplatin. 32. The method according to any one of claims 22 to 31 , wherein the body fluid is urine. 32. The method according to any one of claims 22 to 31 , wherein the body fluid is serum. 34. The method according to any one of claims 1 to 33 , wherein the individual is a human. As a biomarker to determine kidney damage or to predict progression of kidney damage
Of a protein selected from the list in the amino acid sequence of SEQ ID NO: 1 include proteins having 100% identity to the protein, or the amino acid sequence of SEQ ID NO: 2 to 14 having at least 80% identity to the use, Use wherein the protein selected includes a protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 1. 36. Use according to claim 35 , wherein the protein is selected from at least one protein selected from a list comprising SEQ ID NOs: 1-14. 36. Use according to claim 35 , wherein the protein is selected from proteins that are at least 80% identical to SEQ ID NO: 1. 38. Use according to claim 37 , wherein the protein is selected from the protein of SEQ ID NO: 1. 36. Use according to claim 35 , wherein the protein of SEQ ID NO: 5 is selected. 40. Use according to any one of claims 35 to 39 , wherein the renal injury is acute renal failure. 41. Use according to any one of claims 35 to 40 , wherein the renal injury or progression of renal injury is due to administration of at least one nephrotoxic factor. 42. Use according to claim 41 , wherein the nephrotoxic factor is an aminoglycoside antibiotic. 42. Use according to claim 41 , wherein the nephrotoxic factor is cisplatin. A kit comprising at least one probe, the probe comprising:
At least one or any thereof of a protein selected from the list in the amino acid sequence of SEQ ID NO: 1 includes a protein having at least 80% identity with the a protein, or 100% identity to the amino acid sequence of SEQ ID NO: 2 to 14 A kit for recognizing a combination of the above, for determining renal damage, or for predicting progression of renal damage, wherein the selected protein is a protein having at least 80% identity to the amino acid sequence of SEQ ID NO: 1 Contains at least a kit. 45. The kit of claim 44 , wherein the probe recognizes at least one protein selected from the list comprising SEQ ID NOs: 1-14, or a combination thereof. 45. The kit of claim 44 , wherein the probe recognizes a protein that is at least 80% identical to SEQ ID NO: 1. 47. The kit of claim 46 , wherein the probe recognizes SEQ ID NO: 1. 45. The kit of claim 44 , wherein the probe recognizes SEQ ID NO: 5. 49. The kit according to any one of claims 44 to 48 , wherein the probe is linked to a solid support. The kit according to any one of claims 44 to 49 , wherein the probe is an antibody that recognizes the protein. 51. Use of a kit according to any one of claims 44 to 47 , 49 and 50 for determining renal damage or for predicting progression of renal damage in an isolated sample from an individual. In isolated sample from an individual, to determine the risk of kidney damage develops, for determining renal damage, or to predict the progression of renal damage, any one of claims 48-50 Use of the kit described in 1.